pHLARE: a new biosensor reveals decreased lysosome pH in cancer cells.
pHLARE: a new biosensor reveals decreased lysosome pH in cancer cells.
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DOI：
10.1091/mbc.e20-06-0383
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发表时间：
2021-01-15
影响因子：
3.3
通讯作者：
Barber DL
中科院分区：
文献类型：
--
作者：
Webb BA;Aloisio FM;Charafeddine RA;Cook J;Wittmann T;Barber DL
许多溶酶体的功能是由腔内pH值为1.50决定的，包括常驻酸活化水解酶的活性。溶酶体pH（pHlys）通常在神经退行性疾病中增加，并且预测在癌症中降低，使其成为限制这些疾病进展的治疗剂的潜在靶点。然而，准确测量pHlys受到目前使用的染料的限制，这些染料在多个细胞内隔室中积累，并且不能在克隆细胞中繁殖用于纵向研究或用于体内测定。为了解决这一问题，我们开发了一种基因编码的比率式pHlys生物传感器，pHLARE（pH溶酶体活性报告），它主要定位于溶酶体，具有pH 4.0至6.5的动态范围，并可以在细胞中稳定表达。使用pHLARE，我们显示具有雷帕霉素复合物1（mTORC 1）的哺乳动物靶标的抑制活性的降低的pHlys。此外，来自不同组织来源的癌细胞具有比未转化细胞低的pHlys，并且在未转化细胞中稳定表达致癌RasV12足以降低pHlys。pHLARE是一种精确测量pHlys的新工具，用于改善对溶酶体动力学的理解，溶酶体动力学越来越多地被认为是治疗靶点。
Many lysosome functions are determined by a lumenal pH of ∼5.0, including the activity of resident acid-activated hydrolases. Lysosome pH (pHlys) is often increased in neurodegenerative disorders and predicted to be decreased in cancers, making it a potential target for therapeutics to limit the progression of these diseases. Accurately measuring pHlys, however, is limited by currently used dyes that accumulate in multiple intracellular compartments and cannot be propagated in clonal cells for longitudinal studies or used for in vivo determinations. To resolve this limitation, we developed a genetically encoded ratiometric pHlys biosensor, pHLARE (pH Lysosomal Activity REporter), which localizes predominantly in lysosomes, has a dynamic range of pH 4.0 to 6.5, and can be stably expressed in cells. Using pHLARE we show decreased pHlys with inhibiting activity of the mammalian target of rapamycin complex 1 (mTORC1). Also, cancer cells from different tissue origins have a lower pHlys than untransformed cells, and stably expressing oncogenic RasV12 in untransformed cells is sufficient to decrease pHlys. pHLARE is a new tool to accurately measure pHlys for improved understanding of lysosome dynamics, which is increasingly considered a therapeutic target.